Peach cold resistance key gene ppgt5 and application thereof in peach cold resistance breeding

Abstract

The application discloses a peach cold-resistance key gene PpGT5 and application thereof in peach cold-resistance breeding, and relates to the field of plant genetic engineering.The CDS sequence of the gene is shown as SEQ ID NO:1, and the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO:2. PpGT5 The gene is significantly up-regulated in expression under low-temperature induction in peach varieties with strong cold resistance, and the promoter region of the gene contains a low-temperature response element. PpGT5 After overexpression of the gene in tobacco, the cold resistance of the transgenic plants is significantly enhanced, which is manifested in the following aspects: the seed germination rate is increased under low temperature, wilting of leaves is reduced, the growth amount is increased, the contents of soluble protein and soluble sugar are increased, the activities of SOD and POD are enhanced, and the accumulations of MDA and H2O2 are reduced. The application provides a key gene resource for peach cold-resistance molecular breeding, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and specifically relates to a key gene for cold resistance in peaches. PpGT5 And its application in cold-resistant peach breeding. Background Technology

[0002] Peach( Prunus persica Peach (L.) Batsch is an important economic forest tree species in my country, playing a vital role in agricultural production due to its excellent fruit quality and significant economic benefits. In recent years, frequent extreme weather events, especially periodic low-temperature freezing damage, have severely threatened the growth, development, and daily production of peach trees in major northern peach-producing areas (such as Hebei and Shandong provinces), often leading to a sharp decline in yield and even plant death. This has become one of the key bottlenecks restricting the sustainable development of the peach industry. The growing environment is crucial for plant growth and development. Under extreme weather conditions, abiotic stresses such as drought and low temperatures can have a severe impact. When faced with these unfavorable growing environments, plants often need to adapt to environmental changes by altering their growth state and regulating metabolic functions and mechanisms in order to survive. Therefore, exploring the cold-resistant gene resources inherent in peaches, deeply analyzing their molecular mechanisms of response to low-temperature stress, and applying them to cold-resistant molecular breeding is a key approach to fundamentally solving this problem.

[0003] Low-temperature stress can severely damage plant cells, inhibiting plant growth, development, and biomass accumulation. Glycosyltransferases are a class of enzymes widely found in plant cells. They modify glycoproteins or lipids on the cell membrane through glycosylation, increasing cell membrane stability and resistance to damage, playing a crucial role in plant responses to abiotic stress. Members of this family have been extensively studied and identified in multiple species, including Arabidopsis thaliana, soybean, cotton, and tea. Studies have shown that glycosyltransferases not only regulate flowering, control fruit ripening, and promote the synthesis of secondary metabolites such as anthocyanins, but also perform multiple key functions in plant responses to abiotic stress. For example, compared to the wild type, transgenic overexpression… tUGT75D1 Arabidopsis seedlings exhibited phenotypic traits such as small cotyledons and high germination rate; under low temperature conditions, CcUGT110 The gene was expressed at elevated levels in the roots of pigeon pea plants, suggesting that it may play a positive regulatory role in plant cold resistance; while its expression level was inhibited in tea plants. UGT1 The expression of this gene actually reduced damage to tea trees and enhanced their cold resistance, suggesting that this gene may negatively regulate the cold resistance of tea trees. This demonstrates the complexity and diversity of the functions of the glycosyltransferase family members in the plant cold resistance regulatory network, exhibiting both positive and negative regulatory factors.

[0004] Galactosyltransferases are a specific group of members of the glycosyltransferase family, participating in secondary metabolic processes in plants and performing multiple key functions in regulating plant growth and development and responding to abiotic stresses. Despite the diverse functions of the glycosyltransferase family, research on the cold-resistance function of galactosyltransferase genes in peaches under low-temperature stress and their involvement in stress response mechanisms is scarce, resulting in a lack of clear functional gene targets and theoretical basis for molecular breeding of peaches for cold resistance. In previous studies, our research group successfully screened a differentially expressed galactosyltransferase gene from peach materials under low-temperature stress using high-throughput transcriptome sequencing technology, naming it... PpGT5 Preliminary studies have found that under -20℃ low-temperature stress, the relative expression level of this gene was significantly higher in cold-resistant peach varieties than in poorly cold-resistant varieties. This phenomenon suggests... PpGT5 This gene may be an important candidate gene involved in regulating cold resistance in peaches. However, the complete molecular characteristics of this gene, the clear relationship between its expression pattern and the cold resistance of varieties, and whether it can indeed directly confer cold resistance to plants are all unclear. Therefore, cloning... PpGT5 The study identified genes and systematically analyzed their functions in the process of cold resistance in peaches, which has important theoretical and practical significance for promoting molecular breeding of cold-resistant peaches. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a key gene for peach cold resistance. PpGT5 This invention also explores its application in peach cold-resistance breeding. Currently, there are few reports on the cold-resistance function of galactosyltransferase genes in peach under low-temperature stress and whether they participate in the stress response mechanism, resulting in a lack of clear functional gene targets and theoretical basis for molecular breeding of peach cold resistance. This invention is the first to clone and identify a galactosyltransferase gene that positively regulates plant cold resistance from peach. PpGT5 This provides important genetic resources and theoretical basis for cultivating new peach varieties with high cold resistance.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: An isolated nucleic acid molecule encoding a peach galactosyltransferase PpGT5 protein, the amino acid sequence of which is shown in SEQ ID NO:2, or a protein having at least 90% sequence identity with SEQ ID NO:2 and having the function of enhancing plant cold resistance.

[0007] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:1. This nucleic acid molecule is the peach galactosyltransferase gene. PpGT5 The CDS sequence is 1149 bp in length and encodes 382 amino acids.

[0008] The present invention also provides a protein, the amino acid sequence of which is shown in SEQ ID NO:2, or encoded by the aforementioned nucleic acid molecule. The protein is peach galactosyltransferase PpGT5, with a theoretical isoelectric point of 7.10 and a molecular weight of 42.28 kD. It possesses 4–5 transmembrane domains and exhibits a typical multi-transmembrane topology. Subcellular localization of this protein suggests that it may be distributed in the cytoplasm or anchored to the extracellular membrane, potentially playing a crucial role in transmembrane signal transduction, ion or molecular transport.

[0009] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules. In one specific embodiment of the present invention, the recombinant expression vector is obtained by... PpGT5 The gene's CDS sequence was obtained by inserting it into the CAM-FLAG-GFP vector, using EcoRI and XhoI restriction enzyme sites.

[0010] The present invention also provides a host cell comprising the above-described recombinant expression vector. In one specific embodiment of the present invention, the host cell is an Agrobacterium cell.

[0011] The present invention also provides a method for preparing transgenic plants, comprising introducing the above-mentioned nucleic acid molecules or the above-mentioned recombinant expression vector into a target plant to obtain transgenic plants, wherein the transgenic plants exhibit enhanced cold resistance compared with wild types.

[0012] The present invention also provides a method for improving the cold resistance of plants, comprising introducing the above-mentioned nucleic acid molecules into a target plant and overexpressing the nucleic acid molecules in the target plant.

[0013] In the above method, the target plant can be a peach ( Prunus persica ) or tobacco ( Nicotiana tabacum (It can also be other crops or fruit trees with economic value.)

[0014] This invention also provides the application of the above-mentioned nucleic acid molecules, proteins, or recombinant expression vectors in peach cold-resistance breeding. By... PpGT5 Introducing and overexpressing genes into peach varieties with weak cold resistance can significantly improve the cold resistance of the recipient varieties, providing an effective molecular breeding method for cultivating new peach varieties with high cold resistance.

[0015] This invention also provides a method for screening cold-resistant peach varieties, including detecting the expression levels of the aforementioned nucleic acid molecules in peach samples, wherein peach samples with significantly upregulated expression levels under low-temperature stress conditions are cold-resistant peach varieties. In a specific embodiment of this invention, the low-temperature stress condition is a treatment at -20°C. Among cold-resistant peach varieties (such as 'Donghe No. 1', 'Hunchun', and 'Qiuyan'), PpGT5The expression level of the gene increased significantly after treatment at -20℃, while the expression level did not change significantly in varieties with weak cold resistance (such as 'Jin'ao' and 'Jinhuangjin'). Therefore, the expression level of this gene can be used as a molecular marker for screening cold-resistant peach varieties.

[0016] The beneficial effects of this invention are as follows: This invention is the first to clone and identify a galactosyltransferase gene from peach. PpGT5 Furthermore, the positive regulatory function of this gene in plant cold resistance was clarified. Bioinformatics analysis showed that... PpGT5 The promoter region of the gene contains a variety of cis-acting elements related to abiotic stress, such as low-temperature response elements, drought-inducible elements, and abscisic acid response elements, suggesting that the gene plays an important role in the stress response regulatory network.

[0017] Among peach varieties with different cold resistance PpGT5 The expression pattern of this gene is positively correlated with the cold resistance of the variety, indicating that the expression level of this gene can be used as a molecular indicator for identifying the cold resistance of peaches and can be applied to the early screening of cold-resistant germplasm resources.

[0018] By overexpressing in tobacco PpGT5 The transgenic plants exhibited significantly enhanced cold tolerance under low-temperature stress: seed germination rate was significantly improved, leaf wilting was significantly reduced, and plant height and leaf area growth were significantly increased. Physiological indicators showed that overexpression... PpGT5 It can significantly increase the content of soluble protein and soluble sugar in transgenic plants, enhance the activity of superoxide dismutase and peroxidase, and significantly reduce the accumulation of malondialdehyde and hydrogen peroxide, effectively maintaining the balance of reactive oxygen species metabolism in plants under low temperature stress and mitigating the degree of oxidative damage to cell membranes. These results fully demonstrate... PpGT5 It is a key gene in the cold resistance regulation pathway of peach, which can positively regulate the plant's low temperature tolerance and has important application value in the molecular breeding of peach cold resistance. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 For peach PpGT5 Homologous sequence analysis diagram among different species.

[0021] Figure 2 for PpGT5Expression level analysis in primary branches of different peach varieties under low temperature stress.

[0022] Figure 3 This diagram illustrates the genetic transformation process of tobacco plants. A: Aseptic seedling culture; B: Callus tissue formation; C: Generation of resistant shoots; D: Rooting culture; E: Transplanting; F: Flowering; G: Seed harvesting.

[0023] Figure 4 This image shows the PCR positive detection results for T2 generation tobacco plants. In the graph, M: DNA marker; P: control; N: negative control; CK: control plants; 1-18: transgenic plants.

[0024] Figure 5 This is a graph showing the RT-qPCR quantitative analysis of T2 generation transgenic tobacco plants. WT: wild type; OE1-OE18: transgenic plants.

[0025] Figure 6 For low-temperature treatment of genetically modified organisms (GMOs) PpGT5 The effect of -OE (germination rate of wild-type tobacco seeds) on the germination rate of wild-type tobacco seeds.

[0026] Figure 7 For tobacco transgenic lines ( PpGT5 Phenotypic analysis of OE and wild-type (WT) before and after low-temperature treatment.

[0027] Figure 8 The diagram shows the physiological and biochemical responses of WT, OE2, and OE12 plants under ambient and low temperatures. In the diagram, A represents proline content; B represents malondialdehyde content; C represents soluble protein content; D represents soluble sugar content; E represents superoxide dismutase activity; and F represents peroxidase activity.

[0028] Figure 9 Transgenic organisms before and after low-temperature treatment ( PpGT5 Differences in H2O2 content between -OE and wild-type (WT) tobacco leaves are shown in the figure. A: DAB staining results; B: H2O2 content determination results. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0031] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0032] Example 1 This embodiment provides the peach galactosyltransferase gene. PpGT5 Cloning of the compound and its application in cold resistance research.

[0033] 1. Materials and Methods 1.1 Test Materials The experimental site was located at the Changli Fruit Tree Research Institute of the Hebei Academy of Agricultural and Forestry Sciences, and the experimental period covered March 2024 to March 2025. The five peach varieties used in the experiment—'Jin'ao', 'Jinhuangjin', 'Qiuyan', 'Donghe No. 1', and 'Hunchun'—were all provided by the Changli Fruit Tree Research Institute of the Hebei Academy of Agricultural and Forestry Sciences. (Note: The last sentence appears to be unrelated and likely refers to tobacco.) Nicotiana tabacum The materials were provided by Shaanxi Borui Biotechnology Co., Ltd. The experimental materials of five peach varieties were subjected to low-temperature treatment (0℃ and -20℃) in an artificial climate chamber. The low-temperature treatment time and process were in accordance with the method of Li Jie et al. (Evaluation of cold resistance of 1-year-old saplings of different peach germplasm resources. Agricultural Research in Arid Regions, 2025, 43(4): 22-31.). The materials were taken from the first-order branches.

[0034] 1.2 peach PpGT5 Gene cloning and bioinformatics analysis The galactosyltransferase gene PpGT5 It is a low-temperature stress response gene, which was obtained through screening of high-throughput transcriptome data of peach under low-temperature stress. Its NCBI gene sequence number is XM_020563810. PpGT5 Gene cloning primers were designed using Primer Premier 6.0 software (primer sequences are shown in Table 1). RT-qPCR quantification was performed using kits from Novizan Biotechnology Co., Ltd. (Vazyme, Nanjing). First-strand reverse transcription was performed using the HiScript® 11 Q RTSuperMix for qPCR (+gDNA wiper) first-strand synthesis kit (R223-01); quantitative PCR was performed using the ChamQ UniversalSYBR qPCR Master Mix kit (Q711-02). The reaction volume was 10 μL, specifically containing: 1 μL cDNA template, forward and reverse primers (concentration 10 μmol·L⁻¹). -10.5 μL of each of the above-mentioned ingredients, and 8 μL of ddH2O. Amplification was performed on a Roche LightCycler 480 quantitative PCR instrument. The reaction program was: 95℃ for 10 min; followed by 40 cycles, each cycle consisting of 95℃ for 30 s and 60℃ for 1 min. The PCR target fragment was amplified using agarose gel electrophoresis, and the target fragment was recovered according to the instructions of the Magen (Guangzhou) HiPure Gel Pure DNA MiniKit. Positive clones were identified by bacterial culture PCR, and the positive strains were then sent for sequencing.

[0035] The data websites TMHMM-2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ), PeachMD (http: / / www.peachmd.com / # / ), and Expasy-ProtScale (https: / / web.expasy.org / protparam / ) were used to analyze the data. PpGT5 The structure of the gene-encoded protein was predicted, its subcellular localization was analyzed, and its physicochemical properties were determined. The PlantCARE database (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) was used for analysis. PpGT5 Online analysis was performed on potential cis-acting elements of the promoter.

[0036] Table 1 PpGT5 List of gene primers 1.3 peach PpGT5 Gene expression analysis in different cold-resistant varieties The experimental materials consisted of five peach varieties: 'Jin'ao', 'Jinhuangjin', 'Qiuyan', 'Donghe No. 1', and 'Hunchun'. For each variety, 20 one-year-old plants, approximately 1.4m in height and 1.2cm in stem diameter, free from pests and diseases, were selected and subjected to low-temperature treatment (0℃ and -20℃). The low-temperature treatment time and process followed the method of Li Jie et al. (2025). The samples were taken from the first-order branches. After low-temperature treatment, the materials were stored at -80℃ for later use. Total RNA was extracted from each sample according to the instructions of the RNA extraction kit and reverse transcribed into cDNA, using the same method as in section 1.2. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) - Ct Data processing methods.

[0037] 1.4 PpGT5 Construction of overexpression vectors Linearization of the vector CAM-FLAG-GFP: The plasmid CAM-FLAG-GFP was double-digested with EcoRI and XhoI. After digestion, the results were detected by agarose gel electrophoresis. The gel containing the corresponding band of the large fragment of the vector was collected, and the digestion products were recovered using a gel extraction kit from Magen (Guangzhou). Digestion was performed at 37℃ for 40 min, followed by inactivation at 65℃ for 15 min. Ligation of the target fragment with the vector: Reaction buffer was added to a PCR tube, and the target DNA fragment and linearized vector were added at a specific molar ratio. Homologous recombinase was then added, and the mixture was incubated at 50℃ for 15 min. Transformation: A 50 μL tube of competent E. coli cells was thawed on ice, and the cells were resuspended by gently tapping the tube wall. 10 μL of the reaction solution from the previous step was added to the competent cells, and the cells were gently tapped a few times and incubated on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 45 s and quickly placed on ice for 2 min. 700 μL of LB liquid medium was added, and the cells were incubated on a shaker at 37℃ for 60 min. In a clean bench, spread 400 μL of bacterial culture (Str resistance, final concentration 50 mg / L) evenly onto a plate. After the culture is absorbed by the solid medium, incubate overnight at 37°C. Positive clone identification: Colony PCR was used to identify positive clones. Plasmid extraction: Plasmids that were correctly sequenced were extracted using the Plasmid Mini KitⅠ (OMEGA, catalog number: D6943-01).

[0038] 1.5 Genetic transformation of tobacco Select plump, mature tobacco seeds, disinfect them with 75% alcohol for 1 min, then with sodium hypochlorite solution for 10-20 min, rinse four times with sterile water, and inoculate them onto 1 / 2 MS solid medium. Incubate in the dark at 25℃ for 3 days, followed by one week of light incubation. Streak the glycerol bacteria on LB solid medium (containing 100 mg / L spectinomycin or kanamycin, 50 mg / L gentamicin, and 50 mg / L rifampin), incubate at 28℃ for 2 days, and then pick single colonies to inoculate onto LB liquid medium (containing 100 mg / L spectinomycin or kanamycin, 50 mg / L gentamicin, and 50 mg / L rifampin) to prepare an Agrobacterium resuspension with an OD600 of 0.4-1.2. One to two months later, collect sterile, tender leaves, cut them into small pieces, and immerse the cut explants in the Agrobacterium resuspension for 8-10 min. Then, place them on MS medium and incubate in the dark at 25℃ for 2-4 days. After co-culturing, the explants were transferred to MS medium containing resistance to induce shoot formation, with the medium being changed every 15 days. The differentiated resistant shoots were then inoculated onto half MS medium containing resistance, allowing them to develop into complete plantlets. Ntactin -F / R gene primers were used as internal controls to identify transgenic tobacco plants by PCR. High-expression T2 generations were screened for phenotypic analysis.

[0039] 1.6 OverexpressionPpGT5 Analysis of tobacco cold resistance Harvested T2 generation transgenic tobacco seeds and wild-type tobacco seeds were placed in petri dishes under two culture conditions. Normal conditions were: 26℃ light for 16 hours, 26℃ darkness for 8 hours, and relative humidity maintained between 55% and 75%. Low-temperature conditions were: 4℃ light for 16 hours, 4℃ darkness for 8 hours, and relative humidity maintained between 55% and 75%. Seeds were cultured under normal conditions for approximately 7 days, during which rooting and germination were observed. Seeds cultured under 4℃ conditions were also cultured for approximately 7 days, during which rooting and germination were observed. Subsequently, seeds cultured under 4℃ conditions were placed under normal conditions for another 7 days, and germination was observed and recorded, and the germination rate was calculated. The germination rate was calculated using the formula: Seed germination rate (%) = Number of germinated seeds / Total number of seeds × 100%.

[0040] To further determine PpGT5 To investigate the effect on tobacco cold resistance, 20 T2 generation transgenic and 20 wild-type tobacco plants of similar growth status and at 6 weeks of age were selected. Plant height, stem diameter, and leaf area were recorded at this stage. The plants were then placed in an artificial climate chamber at 4℃ (16 hours of light, 8 hours of darkness, 55%-75% relative humidity) for 10 days. Plant height, stem diameter, and leaf area were again measured, and the degree of leaf wilting was observed. Leaf tissue was collected for various index measurements. Plant height and stem diameter were measured using a ruler and vernier calipers, respectively. Leaf area was analyzed using the Wanshen LA-S plant leaf area analysis system. Specific indicators measured included: proline (Pro), malondialdehyde (MDA), soluble sugar, soluble protein content, and superoxide dismutase (SOD) and peroxidase (POD) activities. These indicators were measured according to the instructions of the test kits from Solarbio (Beijing). The qualitative and quantitative analysis methods for reactive oxygen species (ROS) in tobacco plant leaves before and after low temperature stress treatment are as follows: (1) Qualitative analysis: H2O2 in fresh plant leaves was detected by diaminobenzidine (DAB) staining method; (2) Quantitative analysis: H2O2 content was determined according to the instructions of the detection kit from Servicebio (Wuhan). All experiments included control groups and were repeated three times.

[0041] 1.7 Data Processing Data were analyzed using SPSS 26.0 software. The expression levels of the gene in different varieties and under different treatment temperatures, as well as the seed germination rate and physiological indicators of transgenic plants under different treatment temperatures, were analyzed using one-way ANOVA (P < 0.05). The expression level of the gene in T2 generation transgenic tobacco was analyzed using a t-test, with significance markers: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***). Correlation analysis and plotting were performed using Origin 2021 and MEGA 7 software.

[0042] 2 Results and Analysis 2.1 peach PpGT5 Bioinformatics analysis of genes right PpGT5 Analysis and prediction of gene-encoded protein information showed that... PpGT5 The full-length gene is 1665 bp, with a CDS sequence of 1149 bp, encoding 382 amino acids. Its theoretical isoelectric point is 7.10, and its molecular weight is 42.28 kD. The PpGT5 protein is composed of three parts: 23.30% alpha helix, 52.88% random coil, and 23.82% extended strand. Subcellular localization of PpGT5 suggests it may be distributed in the cytoplasm or anchored to the outer surface of the cell membrane. This protein possesses 4–5 transmembrane domains, exhibiting a typical multi-transmembrane topology. Its amino acid sequence shows a clear alternation of hydrophobic and hydrophilic regions, a structural pattern consistent with typical characteristics of channel proteins, carriers, or membrane receptors integrated into membranes, indicating that PpGT5 may play a crucial role in transmembrane signal transduction, ion transport, or molecular transport.

[0043] PpGT5 The CDS sequence of the gene, its nucleotide sequence is shown in SEQ ID NO:1, specifically as follows: The amino acid sequence of the PpGT5 protein is shown in SEQ ID NO:2, specifically: MAWLPTIVSMTMIISEAIFLELTISGGVLLEMLFEEFGGRVELAKAYYKALTDSMKKHFNGNGVIASMQHCNDFMYLGCVGDDFWSKTTGVADGTYWLQGCHAVHCAYSSLWMGNIIHPCAEFHAASRAISGGPIYISDSVGKHNFKLLKSLVLPDGSVLRCQHYALPTRDCLFEDPVHDGKTMLKIWNLN KYTGALGLFNCQGGGWCPKSRRNISAPECSKPLTCLSGPKDIEWNGKSPISIKGMNIFAVYMHQQKKLKLLKLSEKVEISLQPFDFKLLTVSPVRVLPKKFIQFAPIGLVNMLNTGGAIQSLEFEDEENSSNLVRIGVKGCGEMSVFASERPSACKIDGEEVKFDFVDKMVTVQVPWPNPSTSTVVDFLL Online analysis of the Plant CARE database revealed that... PpGT5 The promoter region of the gene contains five classes of cis-acting elements (Table 2). Notably, three of these elements are closely related to responses to abiotic stresses such as low temperature and drought: two low-temperature response elements located at 619-625 bp and 1966-1972 bp, one abscisic acid response element located at 832-837 bp, and one drought-inducing element located at 1758-1764 bp. This result indicates that... PpGT5 It may act as an important regulatory factor, regulating the processes of plant low-temperature tolerance and other abiotic stresses.

[0044] Table 2 Peach PpGT5 Predictive analysis of cis-acting elements in gene promoter regions Evolutionary analysis showed that the PpGT5 protein sequence shared high homology (36.3%-90.6%) with galactosyltransferase protein sequences from plants such as apple, Arabidopsis thaliana, and tomato, and also shared homology with sweet cherry ( Prunus avium The amino acid sequence of A0A6P5TFQ8 showed the highest homology, reaching 90.6%. Figure 1 ).

[0045] 2.2 peach [[ID= Gene expression analysis in different cold-resistant peach varieties Wang Zhaoyuan et al. (2022) and Li Jie et al. (2025) conducted cold resistance tests on different peach varieties and found that 'Donghe No. 1' and 'Hunchun' had strong cold resistance, 'Qiuyan' had relatively strong cold resistance, and 'Jin'ao' and 'Jinhuangjin' had weak cold resistance. RT-qPCR results showed that... ​ This expression was present in the primary branches of different cold-resistant peach varieties. For example... ​ As shown, at 0℃, there was no significant difference in gene expression among the five varieties; after treatment at -20℃, the expression levels of 'Donghe No. 1', 'Hunchun', and 'Qiuyan' significantly increased (P < 0.05), while the expression levels of 'Jin'ao' and 'Jinhuangjin' showed no significant difference. These results indicate that... ​ It can respond to low-temperature stress, and the changes in its expression level show different trends in different cold-resistant varieties. In highly cold-resistant varieties, it is significantly upregulated by low temperature, thus indicating that... ​ It responds to low temperature stress treatment, and its expression level is positively correlated with the cold resistance of the variety.

[0046] 2.3 Genetic transformation and expression in transgenic tobacco plants The complete process of obtaining T0 generation transgenic tobacco plants is as follows: ​ As shown, the process includes sterile seedling culture, callus induction, resistant shoot formation, rooting culture, transplanting, flowering, and seed harvesting; T2 generation transgenic tobacco plants were obtained through subculture. At 6 weeks of age, some T2 generation transgenic tobacco plants were tested for PCR positivity. ​ A total of 18 T2 generation transgenic tobacco plants were obtained, numbered OE1-OE18. Subsequently, qRT-PCR was performed on these 18 positive plants to detect their relative expression levels, as shown below. ​ As shown, the expression levels varied among different strains. Two transgenic strains, OE2 and OE12, with relatively high expression levels, were selected for subsequent cold resistance verification experiments.

[0047] 2.4 Effects of Low Temperature Stress on Transgenic Tobacco Plants 2.4.1 Effects of Low Temperature Stress on Seed Germination like ​ As shown, after culturing wild-type and transgenic tobacco seeds under normal conditions for 7 days, both roots and young leaves grew normally, exhibiting good growth, uniformity, and a germination rate of 100%. Conversely, seeds treated at 4℃ for 7 days did not show rooting or germination; subsequently transferred to normal conditions, the seeds began to root and grow leaves, but the germination rate was significantly lower than under normal conditions, with germination rates of 6.25% for wild-type, 92.67% for transgenic lines OE2, and 93.65% for transgenic lines OE12. After low-temperature treatment, ​The germination rate and leaf growth status of the -OE transgenic seeds were significantly better than those of the wild type, with highly significant differences. The results indicate that the germination of wild-type tobacco seeds was significantly inhibited by low temperature, while... ​ Although the OE transgenic tobacco seeds were affected to some extent, they still maintained a high germination ability and growth vigor, indicating that... ​ Overexpression of the gene can significantly enhance the tolerance of tobacco seeds to low temperatures.

[0048] 2.4.2 Changes in growth morphology of transgenic and wild-type tobacco plants under low temperature stress To clarify ​ The function of tobacco plants in low-temperature stress response was investigated by subjecting 6-week-old plants to a 10-day low-temperature treatment at 4°C. ​ As shown, before low-temperature treatment, the growth status of the transgenic lines (OE2, OE12) and the wild-type (WT) plants was basically the same, with no obvious differences observed; after low-temperature treatment, the leaves of both types of plants showed wilting, but to different degrees: the leaves of the wild-type plants showed severe overall wilting, while... ​ The -OE transgenic plants showed good growth, with only slight wilting of the lower leaves. This result indicates that... ​ Overexpression of the gene can significantly improve the tolerance of tobacco plants to low temperature stress.

[0049] As shown in Table 3, under low-temperature stress, the plant height, stem diameter, and leaf area of ​​the transgenic tobacco lines OE2 and OE12 were significantly higher than those of the wild type, but the increase patterns differed between the transgenic and wild types. Regarding plant height and leaf area: the transgenic lines showed more significant increases, with OE12 increasing in plant height by 21.75% and leaf area by 31.60%; and OE2 increasing in plant height by 26.16% and leaf area by 22.14%. Regarding stem diameter: the WT tobacco stem diameter increased by 25.00%, higher than the 10.71% (OE2) and 22.44% (OE12) of the transgenic lines.

[0050] Table 3. Changes in growth indicators of transgenic tobacco plants under low temperature stress 2.4.3 Determination of physiological and biochemical indicators of transgenic and wild-type tobacco leaves under low temperature stress Before low-temperature stress treatment, there were no significant differences in the contents of proline, malondialdehyde, soluble protein, and soluble sugar in the leaves of transgenic and wild-type plants. The results of 4℃ low-temperature treatment showed that, compared with the wild type, transgenic plants exhibited lower levels of proline and malondialdehyde (MDA). ​ (A, B), but it accumulated more soluble protein and soluble sugar ( ​(C, D). Specifically, under low-temperature stress, compared with the wild type, the proline and malondialdehyde (MDA) contents of the transgenic lines decreased by 27.93% and 18.90%, respectively; while the soluble protein and soluble sugar contents increased by 26.69% and 111.16%, respectively. Proline is an important osmotic regulator. This result indicates that the proline accumulation capacity of transgenic plants may be regulated by other compensatory pathways, but it enhances osmotic regulation capacity by significantly increasing soluble protein and soluble sugar levels, thereby enhancing their cold resistance.

[0051] After being subjected to low-temperature stress at 4℃, ​ The activity of the antioxidant enzyme SOD in transgenic plants was significantly higher than that in wild-type plants. ​ The expression level (E) was increased by 156.60% compared to the wild type, indicating that low temperature can induce overexpression. ​ The significant increase in SOD activity in the plant effectively alleviates the oxidative damage caused by low temperature, thereby enhancing the plant's ability to resist low temperature.

[0052] After treatment with 4℃ low-temperature stress, the activity of the antioxidant enzyme POD in wild-type plants did not differ significantly, while ​ The transgenic plants showed a significant increase in POD activity. ​ The expression level (F) was increased by 54.02% compared to the wild type, indicating that low temperature can induce overexpression. ​ The tobacco plants have an enhanced ability to scavenge reactive oxygen species (ROS), thus preventing them from suffering oxidative damage.

[0053] 2.4.4 Differences in H2O2 in leaves of transgenic and wild-type tobacco under low-temperature stress DAB immunohistochemical staining results are as follows: ​ As shown in -A, under normal conditions, the DAB staining results of wild-type and transgenic tobacco were similar, with no obvious browning. However, after treatment at 4℃, the leaves of the two transgenic lines (OE2 and OE12) showed significantly lighter staining than the wild type, with fewer and lighter brown patches. Quantitative analysis results based on the samples are as follows: ​ As shown in Figure B, before low-temperature stress treatment, there was no significant difference in H2O2 accumulation in the leaves of transgenic and wild-type plants. After 4℃ low-temperature stress treatment, the H2O2 accumulation in wild-type tobacco was significantly higher than that in transgenic tobacco, with the latter decreasing by 43.84% compared to the former, consistent with the immunohistochemical staining results. These results indicate that overexpression after low-temperature treatment... ​ It can effectively reduce the accumulation of ROS in the leaves of genetically modified tobacco plants and protect cells from oxidative damage.

[0054] 3. Discussion Galactosyltransferase (GT) participates in regulating plant signaling pathways and maintaining cellular homeostasis through glycosylation, thereby enabling plants to cope with abiotic stresses such as low temperature and drought, and improving plant stress resistance. Studies have confirmed that... ​ Double mutant plants showed a significant decrease in anthocyanin content, while overexpressing Arabidopsis thaliana... ​ and ​ Its anthocyanin accumulation and antioxidant capacity are significantly enhanced, and this pathway is directly regulated by the transcription factor CBF1, ultimately improving the plant's tolerance to low temperature, drought, and salt stress; inhibiting tea tree... ​ Gene expression, firstly, reduces the amount of gene expression transmitted through the body. ​ The accumulation of catalytically generated nerolipids weakens the reactive oxygen species scavenging system in tea plants, ultimately leading to a significant decrease in cold tolerance due to intensified low-temperature-induced oxidative stress. This invention clones a transmembrane protein encoding 382 amino acids from peach, possessing 4–5 transmembrane domains. Its coding sequence is 1665 bp long, with a predicted molecular weight of 42.28 kD and a theoretical isoelectric point of 7.10. Promoter sequence analysis reveals that it contains multiple cis-regulatory elements related to abiotic stress responses.

[0055] To investigate the galactosyltransferase gene ​ The function of real-time quantitative PCR was used to perform the test, and the results showed that after low-temperature treatment, ​ The expression level of the gene was significantly increased in the cold-resistant varieties 'Qiuyan', 'Donghe No. 1', and 'Hunchun', while the expression level of the cold-resistant varieties 'Jin'ao' and 'Jinhuangjin' showed no significant difference. Therefore, it can be inferred that... ​ This gene may be involved in the plant's cold resistance process. Subsequently, overexpression of this gene in tobacco plants showed that... ​ Although the germination of -OE transgenic tobacco seeds was inhibited by low temperature, their germination rate was still significantly higher than that of wild-type seeds, indicating that this gene plays an active regulatory role in the seed germination process. Therefore, it is speculated that this gene may be involved in the seed germination response to low temperature stress, warranting further in-depth research. Low temperature stress significantly affects the phenotypic development and growth vigor of plants. Its direct damage manifests as leaf wilting, chlorosis caused by chlorophyll degradation, slow plant growth, and local tissue necrosis due to cell membrane system damage. Wilting is the most common physiological response of plants to low temperature stress. Under low temperature stress, plants are most prone to wilting, possibly because low temperature significantly inhibits root water absorption and xylem water transport efficiency, while weakening the plant's perception and response to water deficit, leading to stomatal regulation disorder and ultimately severe water imbalance-induced wilting. This study shows that under low temperature stress, compared with wild-type plants, the leaves of transgenic plants showed lower wilting levels; overexpression... ​The transgenic tobacco plants exhibited significantly higher plant height, stem diameter, and leaf area than the wild type, but displayed different growth response patterns. Specifically, the transgenic tobacco plants showed significantly greater increases in plant height and leaf area than the wild type, which may be related to the gene's involvement in regulating cell elongation or enhancing photosynthesis. This differential expression pattern may be potentially associated with specific metabolic pathways activated by plants under low-temperature stress to maintain cell membrane structural integrity.

[0056] The plasma membrane of plant cells is the first line of defense against abiotic stresses, and its stability is directly related to cellular homeostasis. Therefore, the content of malondialdehyde (MDA), the end product of plasma membrane lipid peroxidation, is widely used as an important indicator for assessing the degree of damage to the cell membrane system. Furthermore, when plants are subjected to abiotic stress, cell membrane permeability often increases, leading to the leakage of intracellular substances and affecting normal physiological metabolic functions. To cope with this change, plants often accumulate osmotic regulators such as proline, soluble sugars, and soluble proteins to maintain osmotic balance, thereby enhancing their water-holding capacity and maintaining normal turgor pressure, thus mitigating the damage caused by abiotic stresses. The results of this invention show that, after low-temperature stress treatment, compared with the wild type, overexpression of [a specific substance]... ​ The significantly reduced MDA content in transgenic tobacco plants suggests a reduced degree of damage to the membrane system; the significantly increased content of soluble protein and soluble sugar suggests an increase in bound water content within the plant, maintaining cell turgor pressure. However, one phenomenon warrants our attention: the increased proline content during overexpression... ​ The transgenic tobacco plants were significantly lower than the wild-type plants, suggesting that overexpression... ​ The changes in proline under low-temperature stress may indirectly affect key enzymes involved in proline synthesis or degradation, and multiple metabolic pathways may be involved, such as... ​ Inhibits key enzymes in proline synthesis by diverting glucose metabolism. ​ or ​ Promoting the proline-degrading enzyme ProDH through activation of the ROS signaling pathway requires further investigation. Reactive oxygen species (ROS), as redox substances, play important physiological roles in plant growth and development. Low-temperature stress disrupts the redox balance within plants, leading to increased ROS levels, biofilm damage, slowed growth, and even death. Under stress, plants enhance their resilience by synergistically scavenging accumulated ROS through SOD and POD to maintain ROS metabolic balance and protect cell membranes from oxidative damage. Studies have shown that under low-temperature stress, tobacco plants overexpress [a specific enzyme / product]. ​ The gene can significantly improve seed germination rate, enhance SOD and POD activity, and effectively alleviate H2O2 accumulation, thereby enhancing the plant's low-temperature tolerance; after low-temperature and PEG6000 induction, tomatoes ​The gene expression level in its leaves showed an increasing trend, and the combined increase in SOD and POD activities in the transgenic lines enhanced the plant's tolerance to abiotic stress. In this invention, after low-temperature stress treatment, compared with the wild type, ​ The transgenic tobacco plants showed significantly increased SOD and POD activities, significantly decreased H2O2 accumulation, and lighter DAB staining. This suggests overexpression. ​ It may enhance the low-temperature tolerance of tobacco plants by effectively maintaining the balance of ROS metabolism in plants under low-temperature stress by enhancing the activity of antioxidant enzyme systems.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An isolated nucleic acid molecule, characterized in that, It encodes the peach galactosyltransferase PpGT5 protein, the amino acid sequence of which is shown in SEQ ID NO:2, or a protein that has at least 90% sequence identity with SEQ ID NO:2 and has the function of enhancing plant cold resistance.

2. The nucleic acid molecule according to claim 1, wherein the nucleotide sequence is shown in SEQ ID NO:

1.

3. A protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO:2, or is encoded by the nucleic acid molecule described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 1 or 2.

5. A host cell, characterized in that, It includes the recombinant expression vector as described in claim 4.

6. A method for preparing transgenic plants, characterized in that, This includes introducing the nucleic acid molecule of claim 1 or 2 or the recombinant expression vector of claim 4 into a target plant to obtain a transgenic plant, wherein the transgenic plant exhibits enhanced cold resistance compared to the wild type.

7. A method for improving the cold resistance of plants, characterized in that, This includes introducing the nucleic acid molecule of claim 1 or 2 into a target plant and overexpressing the nucleic acid molecule in the target plant.

8. The method according to claim 6 or 7, characterized in that, The target plant is peach ( Prunus persica ) or tobacco ( Nicotiana tabacum ).

9. The application of the nucleic acid molecule of claim 1 or 2, the protein of claim 3, or the recombinant expression vector of claim 4 in peach cold-resistant breeding.

10. A method for screening cold-resistant peach varieties, characterized in that, This includes detecting the expression level of the nucleic acid molecules described in claim 1 or 2 in peach samples, wherein peach samples with significantly upregulated expression levels under low-temperature stress are cold-resistant peach varieties.

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